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Updated: Jan 6, 2026

Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells
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Momiji Embryonic Stem Cell System Enabling Live Imaging of Random X-Chromosome Inactivation In Vitro During Embryonic

Manami Koshiguchi1,2, Nao Yonezawa3, Yu Hatano3,4

  • 1Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tokyo, Japan.

Methods in Molecular Biology (Clifton, N.J.)
|November 11, 2025
PubMed
Summary
This summary is machine-generated.

Researchers developed a new system to track X-chromosome inactivation (XCI) in female mammals. This method allows for precise, long-term live imaging of XCI dynamics in embryonic stem cells (ESCs).

Keywords:
Cell fate tracingEmbryonic stem cell differentiationEpigeneticsFluorescent reporterLive-cell imagingMomiji ESCSingle-cell tracingSpinning-disk confocal microscopeX-chromosome inactivation (XCI)

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Area of Science:

  • Genetics
  • Developmental Biology
  • Cell Biology

Background:

  • X-chromosome inactivation (XCI) equalizes gene dosage in eutherian mammals.
  • Observing random XCI (rXCI) in vivo is challenging.
  • Conventional embryonic stem cells (ESCs) can lose chromosomes during culture, complicating rXCI studies.

Purpose of the Study:

  • To develop a robust in vitro model for studying XCI dynamics.
  • To enable long-term, single-cell live imaging of rXCI.
  • To overcome limitations of conventional ESCs in XCI research.

Main Methods:

  • Developed the Momiji ESC (version 2) system with distinct X-chromosome reporters and drug-resistance markers.
  • Utilized drug selection prior to differentiation to prevent X-chromosome loss.
  • Employed spinning-disk confocal microscopy with Z-stack acquisition and multi-tile imaging for low-phototoxicity time-lapse observation (up to 7 days).

Main Results:

  • The Momiji ESC system faithfully models rXCI.
  • Enabled long-term single-cell live imaging of XCI onset and progression.
  • Prevented X-chromosome loss during differentiation and culture.

Conclusions:

  • The Momiji ESC system provides a powerful platform for in vitro XCI research.
  • This system allows for detailed visualization and analysis of XCI dynamics.
  • Facilitates a deeper understanding of gene dosage compensation mechanisms.